Transcriptional regulation is a major event in cell differentiation, proliferation, and apoptosis. Transcriptional activation of a set of genes determines cell destination and for this reason transcription is tightly regulated by a variety of factors. One of its regulatory mechanisms involved in the process is an alteration in the tertiary structure of DNA, which affects transcription by modulating the accessibility of transcription factors to their target DNA segments. Nucleosomal integrity is regulated by the acetylation status of the core histones. In a hypoacetylated state, nucleosomes are tightly compacted and thus are nonpermissive for transcription. They are relaxed by acetylation of the core histones, with the result being permissiveness to transcription. The acetylation status of the histones is governed by the balance of the activities of histone acetyl transferase (HAT) and histone deacetylase (HDAC). Recently, HDAC inhibitors have been found to arrest growth and apoptosis in several types of cancer cells, including colon cancer, T-cell lymphoma, and erythroleukemic cells. Given that apoptosis is a crucial factor in detecting cancer progression, HDAC inhibitors are promising reagents for cancer therapy as effective inducers of apoptosis (Koyama, Y., et al., Blood 96 (2000) 1490-1495).
Several structural classes of HDAC inhibitors have been identified and are reviewed in Marks, P. M., et al., Journal of the National Cancer Institute 92 (2000) 1210-1216. More specifically, WO 98/55449 (by The University of Queensland et al, xe2x80x9cHydroxamic Acid Compounds Having Anticancer And Anti-Parasitic Propertiesxe2x80x9d), and U.S. Pat. No. 5,369,108 (by Breslow, R., et al., xe2x80x9cPotent Inducers Of Terminal Differentiation And Methods Of Use Thereofxe2x80x9d) report alkanoyl hydroxamates with HDAC inhibitory activity.
We have now found that certain tricyclic alkylhydroxamate derivatives possess anti-cell-proliferation properties which are more potent than those in the aforementioned references. These properties are due to HDAC inhibition.
According to the invention there is provided a tricyclic alkylhydroxamate derivative of the formula I 
wherein
A denotes a bond, the groups xe2x80x94CH2xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94, xe2x80x94CH2xe2x80x94CH2-, or xe2x80x94NHxe2x80x94COxe2x80x94;
X denotes the group xe2x80x94NR3xe2x80x94, xe2x95x90CO, or xe2x80x94CH(OH,)xe2x80x94;
Y denotes an oxygen atom, a sulfur atom, or the group xe2x80x94NR4xe2x80x94;
Z denotes a straight chain alkylene group comprising 4, 5, 6, 7, or 8 carbon atoms, wherein one CH2 group may be replaced by an oxygen or a sulfur atom, or wherein 2 carbon atoms form a Cxe2x95x90C double bond, and which is either unsubstituted or substituted by one or two substituents selected from (1-4C)alkyl and halogen atoms;
R1 and R2 denote substituents independently selected from a hydrogen atom, halogen atoms, (1-4C)alkyl, trifluoromethyl, hydroxy, (1-4C)alkoxy, benzyloxy, (1-3C)alkylenedioxy, nitro, amino, (1-4C)alkylamino, di[(1-4C)alkyl]-amino, or (1-4C)alkanoylamino groups;
R3 and R4 independently denote hydrogen atoms or (1-4C)alkyl groups;
their enantiomers, diastereoisomers, racemates, salts and mixtures thereof.
A suitable value for a substituent when it is a halogen atom is, for example, fluoro, chloro, bromo and iodo; when it is (1-4C)alkyl is, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl; when it is (1-4C)alkoxy is, for example, methoxy, ethoxy, propoxy, isopropoxy or butoxy; when it is (1-4C)alkylamino is, for example, methylamino, ethylamino or propylamino; when it is di-[(1-4C)alkyl]amino is, for example, dimethylamino, N-ethyl-N-methylamino, diethylamino, N-methyl-N-propylamino or dipropylamino; when it is (1-4C)alkanoylamino is, for example, formylamido, acetamido, propionamido or butyramido; and when it is (1-3C)alkylenedioxy is, for example, methylenedioxy, ethylenedioxy or propylenedioxy.
Preferred tricycles of formula I are dibenzoxepine, dibenzazepine, fluorene or carbazol.
Y is preferred an oxygen atom. Z is a straight chain alkylene group with 4 to 8 carbon atoms, preferably 4 to 7. The chain can be substituted by one or two halogen atoms, preferably chlorine, or a C1-C4-alkyl group, preferably methyl. One xe2x80x94CH2-group of the chain can be replaced by an oxygen or sulfur atom, however, this group should not be the first or last member of the chain. A CH2xe2x80x94CH2-group of the chain can also form a xe2x80x94Hxe2x95x90CH-group.
Preferred compounds of the invention include tricyclic alkylhydroxamate derivatives of the formula I 
wherein
A denotes a bond, the groups xe2x80x94CH2xe2x80x94Oxe2x80x94, or xe2x80x94NHxe2x80x94COxe2x80x94;
X denotes the group xe2x80x94NR3xe2x80x94, xe2x95x90CO, or xe2x80x94CH(OH)xe2x80x94;
Y denotes an oxygen atom, a sulfur atom, or the group xe2x80x94NR4xe2x80x94;
Z denotes a straight chain alkylene group comprising 4, 5, 6, 7, or 8 carbon atoms, wherein one CH2 group may be replaced by an oxygen or a sulfur atom, or wherein 2 carbon atoms form a Cxe2x95x90C double bond, and which is either unsubstituted or substituted by one or two substituents selected from (1-4C)alkyl and halogen atoms;
R1 and R2 denote substituents independently selected from halogen atoms, (1-4C)alkyl, trifluoromethyl, hydroxy, (1-4C)alkoxy, benzyloxy, (1-3C)alkylenedioxy, nitro, amino, (1-4C)alkylamino, di[(1-4C)alkyl]-amino, or (1-4C)alkanoylamino groups;
R3 and R4 independently denote hydrogen atoms or (1-4C)alkyl groups;
their enantiomers, diastereoisomers, racemates, salts and mixtures thereof.
A tricyclic alkylhydroxamate derivative of the formula I, or a pharmaceutically-acceptable salt thereof, may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Such processes, when used to prepare a tricyclic alkylhydroxamate derivative of the formula I, or a pharmaceutically-acceptable salt thereof, are provided as a further feature of the invention and are illustrated by the following representative examples in which, unless otherwise stated, A, X, Y, Z, R1, R2, R3, and R4 have any of the meanings defined hereinbefore. The starting materials may be obtained by standard procedures of organic chemistry. The preparation of such starting materials is described within the accompanying non-limiting examples. Alternatively starting materials are obtainable by analogous procedures to those illustrated which are within the ordinary skills of an organic chemist.
a) One preferred method for the preparation of compounds of the formula I is the deprotection of compounds of the formula II 
wherein R5 is a suitable protecting group. Compounds of the formula II are new and included in the present invention.
Suitable protecting groups are the benzyl-, p-methoxybenzyl-, tert.butyloxy-ccarbonyl-, trityl-, or silyl groups such as the trimethylsilyl- or dimethyl-tert.butylsilyl-group. The reactions carried out depend on the type of the protecting group. When the protecting group is a benzyl- or p-methoxybenzyl group, the reaction carried out is a hydrogenolysis in an inert solvent such as an alcohol like methanol or ethanol, in the presence of a noble metal catalyst such as palladium on a suitable carrier such as carbon, barium sulfate, or barium carbonate, at ambient temperature and pressure. When the protecting group is the tert.butyloxycarbonyl-, trityl-, or a silyl group such as the trimethylsilyl- or dimethyl-tert.butylsilyl-group, the reaction is carried out in the presence of acids at a temperature between xe2x88x9220xc2x0 C. and 60xc2x0 C., preferably between 0xc2x0 C., and ambient temperature. The acid may be a solution of hydrochloric acid in an inert solvent such as diethyl ether or dioxane, or trifluoro acetic acid in dichloromethane. Alternatively, when the protecting group is a silyl group such as the trimethylsilyl or dimethyl-tert.butylsilyl group, the reaction is carried out in the presence of a fluoride source such as sodium fluoride or tetrabutyl ammonium fluoride in an inert solvent such as dichloromethane.
Compounds of the formula II are obtained by the reaction of a tricyclic alkylhydroxamate of the formula III 
with a compound of formula IV
Wxe2x80x94Zxe2x80x94CONHxe2x80x94Oxe2x80x94R5xe2x80x83xe2x80x83(IV)
wherein W is a displaceable group and Z and R5 have the meaning defined hereinbefore, in the absence or presence of a suitable base.
A suitable displaceable group W is, for example, a halogeno, or sulphonyloxy group, for example a chloro, bromo, methanesulphonyloxy or toluene-p-sulphonyloxy group. A suitable base is, for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine, N-methyl-morpholine or diazabicyclo[5.4.0]undec-7-ene, or, for example, an alkali or alkaline earth metal carbonate or hydroxide, for example sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide or potassium hydroxide.
The reaction is conveniently carried out in the presence of a suitable inert solvent or diluent, for example an alkanol or ester such as methanol, ethanol, isopropanol or ethyl acetate, a halogenated solvent such as methylene chloride, chloroform or carbon tetrachloride, an ether such as tetrahydrofuran or 1,4-dioxane, an aromatic solvent such as toluene, or a dipolar aprotic solvent such as N,N-dimethylformamide, N,N-dimethyl-acetamide, N-methylpyrrolidin-2-one or dimethylsulfoxide. The reaction is conveniently carried out at a temperature in the range, for example, 10 to 250xc2x0 C., preferably in the range 40-200xc2x0 C.
The compounds of the general formula III are either commercially available or can be prepared according to the following literature references or in analogous manners. Compounds of the formula III wherein A denotes a bond and X denotes the group NR3xe2x80x94, can be prepared according to the German Patent Application DE 2928483 (Lauer, K., and Kiegel, E.; Boehringer Mannheim GmbH). Compounds of the formula III wherein A denotes a bond, the group xe2x80x94CH2CH2xe2x80x94, or xe2x80x94CH2xe2x80x94Oxe2x80x94 and X denotes the group xe2x95x90CO, can be prepared according to the German Patent Application DE 2208893 (Winter, W., et al.; Boehringer Mannheim GmbH).
Compounds of the formula IV are prepared by reacting compounds of formula Wxe2x80x94Zxe2x80x94COOH (commercially available) with the compounds of the formula H2Nxe2x80x94Oxe2x80x94R5 (commercially available) wherein W, Z, and R5 have the meaning defined hereinbefore. This is a two step reaction. In the first step, the carboxylate becomes activated. This reaction is carried out in an inert solvent or diluent, for example, in dichloromethane, dioxane, or tetrahydrofuran, in the presence of an activating agent. A suitable reactive derivative of an acid is, for example, an acyl halide, for example an acyl chloride formed by the reaction of the acid with an inorganic acid chloride, for example thionyl chloride; a mixed anhydride, for example an anhydride formed by the reaction of the acid and a chloroformate such as isobutyl chloroformate; an active ester, for example an ester formed by the reaction of the acid and a phenol such as pentafluorophenol, an ester such as pentafluorophenyl trifluoroacetate or an alcohol such as methanol, ethanol, isopropanol, butanol or N-hydroxybenzotriazole; an acyl azide, for example an azide formed by the reaction of the acid and an azide such as diphenylphosphoryl azide; an acyl cyanide, for example a cyanide formed by the reaction of the acid and a cyanide such as diethylphosphoryl cyanide; or the product of the reaction of the acid and a carbodiimide such as dicyclohexylcarbodiimide. The reaction is carried out between xe2x88x9230xc2x0 C. and 60xc2x0 C., conveniently at or below 0xc2x0 C. In the second step, hydroxylamine is added to the solution at the temperature used for the activation, and the temperature is slowly adjusted to ambient temperature.
b) Another preferred method for the preparation of compounds of the formula I involves the reaction of compounds of the formula V 
with hydroxylamine. This reaction typically involves a two-step one-pot procedure. In the first step, the carboxylate of the formula V becomes activated. This reaction is carried out in an inert solvent or diluent, for example, in dichloromethane, dioxane, or tetrahydrofuran, in the presence of an activating agent. A suitable reactive derivative of an acid is, for example, an acyl halide, for example an acyl chloride formed by the reaction of the acid with an inorganic acid chloride, for example thionyl chloride; a mixed anhydride, for example an anhydride formed by the reaction of the acid and a chloroformate such as isobutyl chloroformate; an active ester, for example an ester formed by the reaction of the acid and a phenol such as pentafluorophenol, an ester such as pentafluorophenyl trifluoroacetate or an alcohol such as methanol, ethanol, isopropanol, butanol or N-hydroxybenzotriazole; an acyl azide, for example an azide formed by the reaction of the acid and an azide such as diphenylphosphoryl azide; an acyl cyanide, for example a cyanide formed by the reaction of the acid and a cyanide such as diethylphosphoryl cyanide; or the product of the reaction of the acid and a carbodiimide such as dicyclohexylcarbodiimide. The reaction is carried out between xe2x88x9230xc2x0 C. and 60xc2x0 C., conveniently at or below 0xc2x0 C. In the second step, hydroxylamine is added to the solution, at the temperature used for the activation, and the temperature is slowly adjusted to ambient temperature.
Compounds of the formula V are prepared from compounds of the formula VI 
wherein R6 is an alkyl group, for example, a methyl, ethyl, or tert. butyl group or benzyl group, by hydrolysis. The conditions under which the hydrolysis is carried out depend on the nature of the group R6. When R6 is a methyl or ethyl group, the reaction is carried out in the presence of a base, for example, lithium hydroxide, sodium hydroxide, or potassium hydroxide in an inert solvent or diluent, for example, in methanol or ethanol. When R6 is the tert.butyl group, the reaction is carried out in the presence of an acid, for example, a solution of hydrochloric acid in an inert solvent such as diethyl ether or dioxane, or trifluoro acetic acid in dichloromethane. When R6 is the benzyl group, the reaction is carried out by hydrogenolysis in the presence of a noble metal catalyst such as palladium on a suitable carrier, such as carbon.
Compounds of the formula VI are prepared from compounds of the formula III 
by reaction with compounds of the formula VII
Wxe2x80x94Zxe2x80x94COOxe2x80x94R6xe2x80x83xe2x80x83(VII)
in the presence of a suitable base.
A suitable base is, for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine, N-methylmorpholine or diazabicyclo[5.4.0]undec-7-ene, or, for example, an alkali or alkaline earth metal carbonate or hydroxide, for example sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide or potassium hydroxide.
The reaction is conveniently carried out in the presence of a suitable inert solvent or diluent, for example an alkanol or ester such as methanol, ethanol, isopropanol or ethyl acetate, a halogenated solvent such as methylene chloride, chloroform or carbon tetrachloride, an ether such as tetrahydrofuran or 1,4-dioxane, an aromatic solvent such as toluene, or a dipolar aprotic solvent such as N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidin-2-one or dimethylsulfoxide. The reaction is conveniently carried out at a temperature in the range, for example, 10 to 250xc2x0 C., preferably in the range 40-200xc2x0 C.
Compounds of formula VII are commercially available.
c) A third preferred method for the production of compounds of the formula I involves the reaction of compounds of the formula VIII 
wherein R7 is an (1-4C)alkyl group, for example, a methyl or ethyl group, with hydroxylamine in the presence of a suitable base.
The reaction is carried out in an inert solvent or diluent such as methanol or ethanol at temperatures between 0xc2x0 C. and 100xc2x0 C., conveniently at or near ambient temperature, and at a pH between 9 and 11. A suitable base is, for example, an alcoholate, for example, sodium methylate.
d) Those compounds of the formula I wherein one of the substituents is an amino group may be prepared by the reduction of a derivative of the formula I wherein the substituent is a nitro group. The reduction may conveniently be carried out by any of the many procedures known for such a transformation. The reduction may be carried out, for example, by the hydrogenation of a solution of the nitro compound in an inert solvent or diluent as defined hereinbefore in the presence of a suitable metal catalyst such as palladium or platinum. A further suitable reducing agent is, for example, an activated metal such as activated iron (produced by washing iron powder with a dilute solution of an acid such as hydrochloric acid). Thus, for example, the reduction maybe carried out by heating a mixture of the nitro compound and the activated metal in a suitable solvent or diluent such as a mixture of water and an alcohol, for example, methanol or ethanol, to a temperature in the range, for example, 50 to 150xc2x0 C., conveniently at or near 70xc2x0 C.
e) Those compounds of the formula I wherein X denotes the xe2x80x94CH(OH)xe2x80x94 group maybe prepared by the reduction of a derivative of the formula I wherein X denotes the xe2x95x90CO group. The reduction may conveniently be carried out by any of the many procedures known for such a transformation. The reduction may be carried out, for example, by hydrogenation in an inert solvent or diluent as defined hereinbefore in the presence of a suitable metal catalyst such as palladium or platinum, for example, in methanol or ethanol, at a temperature in the range, for example, 0 to 1 00xc2x0 C., conveniently at or near ambient temperature.
f) Those compounds of the formula I wherein one of the substituents is an (1-4C)alkanoylamino group, are prepared by acylation of a derivative of the formula I wherein the substituent is an amino group. A suitable acylating agent is, for example, any agent known in the art for the acylation of amino to acylamino, for example an acyl halide, for example an alkanoyl chloride or bromide, conveniently in the presence of a suitable base, as defined hereinbefore, an alkanoic acid anhydride or mixed anhydride, for example acetic anhydride or the mixed anhydride formed by the reaction of an alkanoic acid and an alkoxycarbonyl halide, for example an alkoxycarbonyl chloride, in the presence of a suitable base as defined hereinbefore. In general the acylation is carried out in a suitable inert solvent or diluent as defined hereinbefore and at a temperature, in the range, for example, xe2x88x9230 to 1 20xc2x0 C., conveniently at or near ambient temperature.
Compounds of the formula VIII are prepared from a compound of the general formula III reacted with a compound of formula Wxe2x80x94Zxe2x80x94COOR7, in like manner to the reaction of a compound of formula III with one of formula VII.
The enantiomers or diastereoisomers of the compounds of formula I can be obtained by usual methods as column chromatography or crystallization or optical resolution of enantiomers by treatment with optically active acids or bases or by using optically active starting materials.
xe2x80x9cPharmaceutically acceptable saltsxe2x80x9d refer to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide. The chemical modification of a pharmaceutical compound (i.e., a drug) into a salt is a technique well known to pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.
According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a tricyclic alkylhydroxamate derivative of the formula I, or enantiomer, diastereoisomer, racemate, pharmaceutically-acceptable salt or mixture thereof, in association with a pharmaceutically-acceptable diluent or carrier. The composition may be in a form suitable for oral administration, for example as a tablet or capsule, for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. In general the above compositions may be prepared in a manner using conventional excipients. The tricyclic alkylhydroxamate will normally be administered to a warm-blooded animal at a unit dose within the range 5-5000 mg per square meter body area of the animal, i.e. approximately 0.1-100 mg/kg , and this normally provides a therapeutically-effective dose. A unit dose form such as a tablet or capsule will usually contain, for example 1-250 mg of active ingredient. Preferably a daily dose in the range of 1-50 mg/kg is employed. However the daily dose will necessarily be varied depending upon the host treated, the particular route of administration, and the severity of the illness being treated. Accordingly the optimum dosage may be determined by the practitioner who is treating any particular patient.
According to a further aspect of the present invention there is provided a tricyclic alkylhydroxamate derivative of the formula I as defined hereinbefore for use in a method of treatment of the human or animal body by therapy. We have now found that the compounds of the present invention possess anti-cell-proliferation properties which are believed to arise from their histone deacetylase inhibitory activity. Accordingly the compounds of the present invention provide a method for treating the proliferation of malignant cells. Accordingly the compounds of the present invention are expected to be useful in the treatment of cancer by providing an anti-proliferative effect, particularly in the treatment of cancers of the breast, lung, colon, rectum, stomach, prostate, bladder, pancreas and ovary. It is in addition expected that a derivative of the present invention will possess activity against a range of leukemias, lymphoid malignancies and solid tumors such as carcinomas and sarcomas in tissues such as the liver, kidney, prostate and pancreas.
Thus according to this aspect of the invention there is provided the use of a tricyclic alkylhydroxamate derivative of the formula I, or a pharmaceutically-acceptable salt thereof, as defined hereinbefore in the manufacture of a medicament for use in the production of an anti-cell-proliferation effect in a warm-blooded animal such as man.
According to a further feature of this aspect of the invention there is provided a method for producing an anti-cell-proliferation effect in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a tricyclic alkylhydroxamate derivative as defined hereinbefore.
The anti-cell-proliferation treatment defined hereinbefore may be applied as a sole therapy or may involve, in addition to the compounds of the invention, one or more other anti-tumor substances, for example those selected from, for example, mitotic inhibitors, for example vinblastine; alkylating agents, for example cis-platin, carboplatin and cyclophosphamide; inhibitors of microtubule assembly, like paclitaxel or other taxanes; antimetabolites, for example 5-fluorouracil, capecitabine, cytosine arabinoside and hydroxyurea, or, for example, intercalating antibiotics, for example adriamycin and bleomycin; immunostimulants, for example trastuzumab; DNA synthesis inhibitors, e.g. gemcitabine; enzymes, for example asparaginase; topoisomerase inhibitors, for example etoposide; biological response modifiers, for example interferon; and anti-hormones, for example antioestrogens such as tamoxifen or, for example antiandrogens such as (4xe2x80x2-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3xe2x80x2-(trifluoromethyl) propionanilide, or other therapeutic agents and principles as described in, for example, Cancer: Principles and Practice of Oncology, Vincent T. DeVita, Jr., Samuel Hellmann, Steven A. Rosenberg; 5th Ed., Lippincott-Raven Publishers, 1997. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of individual components of the treatment. According to this aspect of the invention there is provided a pharmaceutical product comprising a tricyclic alkylhydroxamate derivative of the formula I as defined hereinbefore and an additional anti-tumor substance as defined hereinbefore for the conjoint treatment of cancer.